Schizotrophic Sclerotinia sclerotiorum-Mediated Root and Rhizosphere Microbiome Alterations Activate Growth and Disease Resistance in Wheat

ABSTRACT Sclerotinia sclerotiorum, a widespread pathogen of dicotyledons, can grow endophytically in wheat, providing protection against Fusarium head blight and stripe rust and enhancing wheat yield. In this study, we found that wheat seed treatment with strain DT-8, infected with S. sclerotiorum hypovirulence-associated DNA virus 1 (SsHADV-1) and used as a “plant vaccine” for brassica protection, could significantly increase the diversity of the fungal and bacterial community in rhizosphere soil, while the diversity of the fungal community was obviously decreased in the wheat root. Interestingly, the relative abundance of potential plant growth-promoting rhizobacteria (PGPR) and biocontrol agents increased significantly in the DT-8-treated wheat rhizosphere soil. These data might be responsible for wheat growth promotion and disease resistance. These results may provide novel insights for understanding the interaction between the schizotrophic microorganism and the microbiota of plant roots and rhizosphere, screening and utilizing beneficial microorganisms, and further reducing chemical pesticide utilization and increasing crop productivity. IMPORTANCE Fungal pathogens are seriously threatening food security and natural ecosystems; efficient and environmentally friendly control methods are essential to increase world crop production. S. sclerotiorum, a widespread pathogen of dicotyledons, can grow endophytically in wheat, providing protection against Fusarium head blight and stripe rust and enhancing wheat yield. In this study, we discovered that S. sclerotiorum treatment increased the diversity of the soil fungal and bacterial community in rhizosphere soil, while the diversity of the fungal community was obviously decreased in the wheat root. More importantly, the relative abundance of potential PGPR and bio-control agents increased significantly in the S. sclerotiorum-treated wheat rhizosphere soil. The importance of this work is that schizotrophic S. sclerotiorum promotes wheat growth and enhances resistance against fungal diseases via changes in the structure of the root and rhizosphere microbiome.

2. L200-L203: "a total of 19 differentially abundant fungal genera were observed in wheat roots, including 2 genera with increased abundance and 17 genera had decreased abundance in the root of DT-8-treated wheat." should be modified as "a total of 18 differentially abundant fungal genera were observed in wheat roots, including 2 genera with increased abundance and 16 genera had decreased abundance in the root of DT-8-treated wheat." 3. L203-L204: "Rhizoctonia and Sclerotinia showed higher abundance in the rhizosphere soil of the DT-8-treated wheat, while lower abundance in the control." should be modified as "Rhizoctonia and Sclerotinia showed higher abundance in the root of the DT-8-treated wheat, while lower abundance in the control." 4. L208: "Phaeosphaeria, Enterographa, Podospora, Leptospora and Botrytis ( Fig. 5A and" there is no Podospora in Fig. 5A. 5. L238-L240: "Notably, well-known bio-control microbes, including Brevibacillus, Microbacterium, Paenibacillus, Bacillus, and Lecanicillium are enriched in the root and rhizosphere soil corresponding DT-8-treated wheat." should be modified as "Notably, well-known bio-control microbes, including Brevibacillus, Microbacterium, Paenibacillus, Bacillus, and Lecanicillium are enriched in the rhizosphere soil corresponding DT-8-treated wheat." 6. L241-L263: In this venn figure comparison, the OTUs numbers is inconsistent with the Fig. 6. It should be checked carefully. 7. L283-L284: Where is Supplementary Table 8? 8. L270-L271: "As a result of ITS sequencing, 57 indicator species from the top 600 OTUs are given in (Supplementary Table  7)," is it 57 or 47? Please check carefully. 9. L114-L117: Fig 1D and Fig 1E should be cited in this site.
Reviewer #2 (Comments for the Author): The authors provided a logical and clear report on the rationale of the experiments and their results. The data are also well presented and the results support the authors conclusion. I don't have particular concerns about most of the experiments. To strengthen the manuscript, I suggest the authors present some previous results about the rhizosphere microbiome between mycovirus-mediated hypovirulent strain and non-virus infected strains in the introduction section. If the relevant results obtained previously, non-virus infected strains tested as control should also be discussed in discussion section.

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Comments:
In this study, strain DT-8 could significantly increase the diversity of the fungal and bacterial community in rhizosphere soil, while the diversity of the fungal community was obviously decreased in the wheat root. However, there are some problems in the data in the manuscript. It need more modifications. It could be accepted after revised. Specific commments: 1. L154-L156:"These results indicate that S. sclerotiorum-treatment significantly increased fungal and bacterial diversity index in wheat rhizosphere soil, whereas declining thefungal diversity" should be modified as "These results indicate that S. sclerotiorum-treatment significantly increased fungal and bacterial diversity index in wheat rhizosphere soil, whereas declining thefungal diversity in roots". 2. L200-L203: "a total of 19 differentially abundant fungal genera were observed in wheat roots, including 2 genera with increased abundance and 17 genera had decreased abundance in the root of DT-8-treated wheat." should be modified as "a total of 18 differentially abundant fungal genera were observed in wheat roots, including 2 genera with increased abundance and 16 genera had decreased abundance in the root of DT-8treated wheat." 3. L203-L204: "Rhizoctonia and Sclerotinia showed higher abundance in the rhizosphere soil of the DT-8-treated wheat, while lower abundance in the control." should be modified as "Rhizoctonia and Sclerotinia showed higher abundance in the root of the DT-8-treated wheat, while lower abundance in the control." 4. L208: "Phaeosphaeria, Enterographa, Podospora, Leptospora and Botrytis ( Fig. 5A and" there is no Podospora in Fig. 5A. 5. L238-L240: "Notably, well-known bio-control microbes, including Brevibacillus, Microbacterium, Paenibacillus, Bacillus, and Lecanicillium are enriched in the root and rhizosphere soil corresponding DT-8-treated wheat." should be modified as "Notably, well-known bio-control microbes, including Brevibacillus, Microbacterium, Paenibacillus, Bacillus, and Lecanicillium are enriched in the rhizosphere soil corresponding DT-8-treated wheat." 6. L241-L263: In this venn figure comparison, the OTUs numbers is inconsistent with the Fig. 6. It should be checked carefully. 7. L283-L284: Where is Supplementary Table 8? 8. L270-L271: "As a result of ITS sequencing, 57 indicator species from the top 600 OTUs are given in (Supplementary Table 7)," is it 57 or 47? Please check carefully. 9. L114-L117: Fig 1D and Fig 1E should be cited in this site.

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Response #1: Thank you for processing the manuscript "Spectrum00981-23" and for the reviewer's comments. We have revised the manuscript carefully according to the comments, which we hope will meet with their approval.

Comments from reviewers:
Reviewer #1 (Comments for the Author): In this study, strain DT-8 could significantly increase the diversity of the fungal and bacterial community in rhizosphere soil, while the diversity of the fungal community was obviously decreased in the wheat root.

Response #2:
We are very grateful for these kind comments, and we have now modified the manuscript as appropriate to correct any deficiencies indicated by this referee as follows: Specific commments: 1. L154-L156: "These results indicate that S. sclerotiorum-treatment significantly increased fungal and bacterial diversity index in wheat rhizosphere soil, whereas declining the fungal diversity" should be modified as "These results indicate that S. sclerotiorum-treatment significantly increased fungal and bacterial diversity index in wheat rhizosphere soil, whereas declining the fungal diversity in roots".  In the original manuscript it was probably the limitation of the number of Supplementary Tables that caused Supplementary Table 8 to be lost, we added   Supplementary Table 8 in the revised version. please see Supplementary Table 8. 8. L270-L271: "As a result of ITS sequencing, 57 indicator species from the top 600 OTUs are given in (Supplementary Table 7

Response #12:
We are very grateful for this kind comment. We have now added more information about our previous results about the beneficial effect between mycovirus-mediated hypovirulent strain and non-virus infected strain of S. sclerotiorum on wheat plants in the introduction section, please see line 97-102.
We also added this information in the discussion section in the revised version.
Previously, we demonstrated that the beneficial effect of S. sclerotiorum on wheat was not strain dependent and did not depend on full virulence on susceptible hosts. Furthermore, given the potential associated risks of S. sclerotiorum virulent strain to dicotyledonous host plants such as rapeseed and soybean in natural ecosystems, as well as the stability of the SsHADV-1 in strain DT-8 and its potential as a "plant vaccine" against sclerotinia disease in rapeseed. We only used the mycovirus-mediated hypovirulent DT-8 strain that is safe for dicotyledons such as rapeseed in this study. Therefore, we speculate that there is no significant difference in the effects of the strong virulent strain and the mycovirus-mediated hypovirulent strain on the wheat rhizosphere microbiome. Your manuscript has been accepted, and I am forwarding it to the ASM Journals Department for publication. You will be notified when your proofs are ready to be viewed.
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